Beta-thalassemia - SJSU Computer Science Departmentkhuri/SMPD_287/Take_Home/beta-thalassemia.pdf ·...

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Beta-thalassemia Antonio Cao, MD 1 , and Renzo Galanello, MD 2 TABLE OF CONTENTS Clinical Features .................................................................................................62 Beta-thalassemia major .................................................................................62 Beta-thalassemia intermedia ........................................................................62 Hematological Features .....................................................................................63 Beta-thalassemia carrier state.......................................................................63 Thalassemia major .........................................................................................63 Thalassemia intermedia.................................................................................63 Molecular Genetics ............................................................................................63 Beta-thalassemia .............................................................................................63 Beta-thalassemic hemoglobinopathies .......................................................65 Beta-thalassemia-like mutations mapping outside the beta globin gene cluster ........................................................................................66 Molecular diagnosis of beta-thalassemia ...................................................66 Phenotype-Genotype Correlation ...................................................................66 Homozygous beta-thalassemia ....................................................................66 Heterozygous beta-thalassemia ...................................................................67 Other clinical genetic modifiers...................................................................68 Beta-Thalassemia Carrier Identification ..........................................................68 Carrier detection procedure .........................................................................69 Molecular diagnosis of modifying genes ...................................................69 Population Screening ........................................................................................71 Prenatal diagnosis ..........................................................................................71 Clinical Management ........................................................................................71 Transfusion program .....................................................................................71 Transfusional iron overload ..........................................................................71 Two other chelators have been introduced into clinical use: deferiprone and deferasirox .........................................................................72 Follow-up.........................................................................................................72 Bone marrow transplantation ......................................................................73 Therapies under investigation......................................................................73 Abstract: Beta-thalassemia is caused by the reduced (beta ) or absent (beta 0 ) synthesis of the beta globin chains of the hemoglobin tetramer. Three clinical and hematological conditions of increasing severity are recognized, i.e., the beta-thalassemia carrier state, thalassemia interme- dia, and thalassemia major. The beta-thalassemia carrier state, which results from heterozygosity for beta-thalassemia, is clinically asymp- tomatic and is defined by specific hematological features. Thalassemia major is a severe transfusion-dependent anemia. Thalassemia interme- dia comprehend a clinically and genotypically very heterogeneous group of thalassemia-like disorders, ranging in severity from the asymp- tomatic carrier state to the severe transfusion-dependent type. The clinical severity of beta-thalassemia is related to the extent of imbalance between the alpha and nonalpha globin chains. The beta globin (HBB) gene maps in the short arm of chromosome 11, in a region containing also the delta globin gene, the embryonic epsilon gene, the fetal A- gamma and G-gamma genes, and a pseudogene (B1). Beta-thalasse- mias are heterogeneous at the molecular level. More than 200 disease- causing mutations have been so far identified. The majority of mutations are single nucleotide substitutions, deletions, or insertions of oligonu- cleotides leading to frameshift. Rarely, beta-thalassemia results from gross gene deletion. In addition to the variation of the phenotype resulting from allelic heterogeneity at the beta globin locus, the pheno- type of beta-thalassemia could also be modified by the action of genetic factors mapping outside the globin gene cluster and not influencing the fetal hemoglobin. Among these factors, the ones best delineated so far are those affecting bilirubin, iron, and bone metabolisms. Because of the high carrier rate for HBB mutations in certain populations and the availability of genetic counseling and prenatal diagnosis, population screening is ongoing in several at-risk populations in the Mediterranean. Population screening associated with genetic counseling was extremely useful by allowing couples at risk to make informed decision on their reproductive choices. Clinical management of thalassemia major con- sists in regular long-life red blood cell transfusions and iron chelation therapy to remove iron introduced in excess with transfusions. At present, the only definitive cure is bone marrow transplantation. Ther- apies under investigation are the induction of fetal hemoglobin with pharmacologic compounds and stem cell gene therapy. Genet Med 2010:12(2):61–76. Key Words: beta-thalassemia, thalassemia major, thalassemia inter- media, iron chelation, thalassemia prevention B eta-thalassemia is one of most common autosomal recessive disorders worldwide. High prevalence is present in popula- tions in the Mediterranean, Middle-East, Transcaucasus, Central Asia, Indian subcontinent, and Far East. It is also relatively common in populations of African descent. The highest inci- dences are reported in Cyprus (14%), Sardinia (12%), and South East Asia 1,2 Detailed information on the frequency of beta- thalassemia in the different regions is available from Weatherall and Clegg 1 The high gene frequency of beta-thalassemia in these regions is most likely related to the selective pressure from Plasmodium falciparum malaria, as it is indicated by its distribution quite similar to that of present or past malaria endemia. 3 Carriers of beta-thalassemia are indeed relatively protected against the invasion of Plasmodium falciparum. How- ever, because of population migration and, to a limited extent, slave trade, beta-thalassemia is, at present, also common in Northern Europe, North and South America, Caribbean, and Australia. Beta-thalassemia is caused by the reduced (beta ) or absent (beta 0 ) synthesis of the beta globin chains of the hemoglobin (Hb) tetramer, which is made up of two alpha globin and two beta globin chains (alpha 2 beta 2 ). 1 Three clinical and hemato- logical conditions of increasing severity are recognized, i.e., the From the 1 Istituto di Neurogenetica e Neurofarmacologia, Consiglio Nazio- nale delle Ricerche, Cagliari, Italy; and 2 Clinica Pediatrica and Dipartimento di Scienze Biomediche e Biotecnologie, Universita ` di Cagliari, Ospedale Regionale Microcitemie ASL Cagliari, Cagliari, Italy. Antonio Cao, MD, Ospedale Regionale Microcitemie, Via Jenner s/n, 09121 Cagliari, Italy. E-mail: [email protected]. Disclosure: The authors declare no conflict of interest. Submitted for publication August 14, 2009. Accepted for publication November 1, 2009. Published online ahead of print January 21, 2010. DOI: 10.1097/GIM.0b013e3181cd68ed GENETEST REVIEW Genetics in Medicine Genetics IN Medicine • Volume 12, Number 2, February 2010 61

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Beta-thalassemiaAntonio Cao, MD1, and Renzo Galanello, MD2

TABLE OF CONTENTS

Clinical Features .................................................................................................62Beta-thalassemia major .................................................................................62Beta-thalassemia intermedia ........................................................................62

Hematological Features.....................................................................................63Beta-thalassemia carrier state.......................................................................63Thalassemia major .........................................................................................63Thalassemia intermedia.................................................................................63

Molecular Genetics ............................................................................................63Beta-thalassemia.............................................................................................63Beta-thalassemic hemoglobinopathies .......................................................65Beta-thalassemia-like mutations mapping outside the betaglobin gene cluster ........................................................................................66Molecular diagnosis of beta-thalassemia ...................................................66

Phenotype-Genotype Correlation ...................................................................66Homozygous beta-thalassemia ....................................................................66

Heterozygous beta-thalassemia ...................................................................67Other clinical genetic modifiers...................................................................68

Beta-Thalassemia Carrier Identification ..........................................................68Carrier detection procedure.........................................................................69Molecular diagnosis of modifying genes ...................................................69

Population Screening ........................................................................................71Prenatal diagnosis ..........................................................................................71

Clinical Management ........................................................................................71Transfusion program .....................................................................................71Transfusional iron overload ..........................................................................71Two other chelators have been introduced into clinical use:deferiprone and deferasirox .........................................................................72Follow-up.........................................................................................................72Bone marrow transplantation ......................................................................73Therapies under investigation......................................................................73

Abstract: Beta-thalassemia is caused by the reduced (beta�) or absent(beta0) synthesis of the beta globin chains of the hemoglobin tetramer.Three clinical and hematological conditions of increasing severity arerecognized, i.e., the beta-thalassemia carrier state, thalassemia interme-dia, and thalassemia major. The beta-thalassemia carrier state, whichresults from heterozygosity for beta-thalassemia, is clinically asymp-tomatic and is defined by specific hematological features. Thalassemiamajor is a severe transfusion-dependent anemia. Thalassemia interme-dia comprehend a clinically and genotypically very heterogeneousgroup of thalassemia-like disorders, ranging in severity from the asymp-tomatic carrier state to the severe transfusion-dependent type. Theclinical severity of beta-thalassemia is related to the extent of imbalancebetween the alpha and nonalpha globin chains. The beta globin (HBB)gene maps in the short arm of chromosome 11, in a region containingalso the delta globin gene, the embryonic epsilon gene, the fetal A-gamma and G-gamma genes, and a pseudogene (�B1). Beta-thalasse-mias are heterogeneous at the molecular level. More than 200 disease-causing mutations have been so far identified. The majority of mutationsare single nucleotide substitutions, deletions, or insertions of oligonu-cleotides leading to frameshift. Rarely, beta-thalassemia results fromgross gene deletion. In addition to the variation of the phenotyperesulting from allelic heterogeneity at the beta globin locus, the pheno-type of beta-thalassemia could also be modified by the action of geneticfactors mapping outside the globin gene cluster and not influencing thefetal hemoglobin. Among these factors, the ones best delineated so farare those affecting bilirubin, iron, and bone metabolisms. Because of the

high carrier rate for HBB mutations in certain populations and theavailability of genetic counseling and prenatal diagnosis, populationscreening is ongoing in several at-risk populations in the Mediterranean.Population screening associated with genetic counseling was extremelyuseful by allowing couples at risk to make informed decision on theirreproductive choices. Clinical management of thalassemia major con-sists in regular long-life red blood cell transfusions and iron chelationtherapy to remove iron introduced in excess with transfusions. Atpresent, the only definitive cure is bone marrow transplantation. Ther-apies under investigation are the induction of fetal hemoglobin withpharmacologic compounds and stem cell gene therapy. Genet Med2010:12(2):61–76.

Key Words: beta-thalassemia, thalassemia major, thalassemia inter-media, iron chelation, thalassemia prevention

Beta-thalassemia is one of most common autosomal recessivedisorders worldwide. High prevalence is present in popula-

tions in the Mediterranean, Middle-East, Transcaucasus, CentralAsia, Indian subcontinent, and Far East. It is also relativelycommon in populations of African descent. The highest inci-dences are reported in Cyprus (14%), Sardinia (12%), and SouthEast Asia1,2 Detailed information on the frequency of beta-thalassemia in the different regions is available from Weatheralland Clegg1 The high gene frequency of beta-thalassemia inthese regions is most likely related to the selective pressurefrom Plasmodium falciparum malaria, as it is indicated by itsdistribution quite similar to that of present or past malariaendemia.3 Carriers of beta-thalassemia are indeed relativelyprotected against the invasion of Plasmodium falciparum. How-ever, because of population migration and, to a limited extent,slave trade, beta-thalassemia is, at present, also common inNorthern Europe, North and South America, Caribbean, andAustralia.

Beta-thalassemia is caused by the reduced (beta�) or absent(beta0) synthesis of the beta globin chains of the hemoglobin(Hb) tetramer, which is made up of two alpha globin and twobeta globin chains (alpha2beta2).1 Three clinical and hemato-logical conditions of increasing severity are recognized, i.e., the

From the 1Istituto di Neurogenetica e Neurofarmacologia, Consiglio Nazio-nale delle Ricerche, Cagliari, Italy; and 2Clinica Pediatrica and Dipartimentodi Scienze Biomediche e Biotecnologie, Universita di Cagliari, OspedaleRegionale Microcitemie ASL Cagliari, Cagliari, Italy.

Antonio Cao, MD, Ospedale Regionale Microcitemie, Via Jenner s/n, 09121Cagliari, Italy. E-mail: [email protected].

Disclosure: The authors declare no conflict of interest.

Submitted for publication August 14, 2009.

Accepted for publication November 1, 2009.

Published online ahead of print January 21, 2010.

DOI: 10.1097/GIM.0b013e3181cd68ed

GENETEST REVIEW Genetics in Medicine

Genetics IN Medicine • Volume 12, Number 2, February 2010 61

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beta-thalassemia carrier state, thalassemia intermedia, andthalassemia major. The beta-thalassemia carrier state, whichresults from heterozygosity for beta-thalassemia, is clinicallyasymptomatic and is defined by specific hematological features.Thalassemia major is a severe transfusion-dependent anemia.Thalassemia intermedia comprehends a clinically and genotyp-ically very heterogeneous group of thalassemia-like disorders,ranging in severity from the asymptomatic carrier state to thesevere transfusion-dependent type. The clinical severity of beta-thalassemia is related to the extent of imbalance between thealpha globin and nonalpha globin chains. The nonalpha globinchains include, in addition to the beta globin chains, also thegamma chains, which are a specific component of fetal Hb(HbF; alpha2gamma2) and are present in a small amount innormal adult individuals and in increased but variable amount inthe beta-thalassemia syndromes. Within the red blood cell pre-cursors, when the beta globin chains are reduced or absent, theunassembled alpha chains precipitate and lead to oxidativedamage of the cell membrane, thereby resulting in apoptosis(ineffective erythropoiesis).4–6

CLINICAL FEATURES

Beta-thalassemia majorHomozygotes for beta-thalassemia may develop either

thalassemia major or thalassemia intermedia. Individuals withthalassemia major usually come to medical attention within thefirst 2 years and require regular blood transfusion to survive.Those presenting later do not require transfusion and receive adiagnosis of thalassemia intermedia. Differentiation of thalas-semia major from thalassemia intermedia at presentation is adifficult and critical issue that should be strongly pursued,because it may avoid unnecessary transfusions in thalassemiaintermedia and start early transfusions in thalassemia major.Analysis of the genotype at the alpha and beta loci and testingfor the presence of ameliorating genetic factors may be useful inthis differentiation (see later).

Affected infants with thalassemia major fail to thrive andbecome progressively pale. Feeding problems, diarrhea, irrita-bility, recurrent bouts of fever, and enlargement of the abdo-men, caused by splenomegaly, may occur. If a regular transfu-sion program that maintains a minimum Hb concentration of95–105 g/L is initiated, then growth and development are nor-mal until the age of 10–11 years. After the age of 10–11 years,affected individuals are at risk of developing severe complica-tions related to posttransfusional iron overload, depending ontheir compliance with chelation therapy.

Complications of iron overload include growth retardationand failure of sexual maturation and also those complicationsobserved in adults with HFE-associated hereditary hemochro-matosis (HH): involvement of the heart (dilated myocardiopa-thy and pericarditis), liver (chronic hepatitis, fibrosis, and cir-rhosis), and endocrine glands (resulting in diabetes mellitus andinsufficiency of the parathyroid, thyroid, pituitary, and, lesscommonly, adrenal glands). Infectious complications, includinghepatitis B and C virus and HIV, relatively common in oldpatients, are now very rare because of the introduction ofvaccination (hepatitis B) and development of blood donorscreening methods based on viral nucleic acid enzymatic am-plification. Other complications are hypersplenism (usually re-lated to late and irregular transfusions), venous thrombosis(occurring especially after splenectomy), osteoporosis (which ismultifactorial, being strongly associated with bone marrow ex-pansion, hypogonadism, diabetes mellitus, hypothyroidism, hy-

poparathyroidism, low insulin-like growth factor 1, cardiac dys-function, and may regard specific patients because of thepresence of peculiar genetic predisposing factors; see later), andlung hypertension (secondary to chronic hemolysis).7–9 The riskfor hepatocellular carcinoma is increased because of liver viralinfection, iron overload, and longer survival.10

Survival of individuals who have been well transfused andtreated with appropriate chelation extends beyond the age of 30years. Myocardial disease caused by transfusional siderosis isthe most important life-limiting complication of iron overloadin beta-thalassemia. In fact, cardiac complications are reportedto cause 71% of deaths in individuals with beta-thalassemiamajor.11

The classic clinical picture of thalassemia major is currentlyonly seen in some developing countries, in which the resourcesfor carrying out long-term transfusion programs are not avail-able. The most relevant features of untreated or poorly trans-fused individuals are growth retardation, pallor, jaundice, brownpigmentation of the skin, poor musculature, genu valgum, hep-atosplenomegaly, leg ulcers, development of masses from ex-tramedullary hematopoiesis, and skeletal changes that resultfrom expansion of the bone marrow. These skeletal changesinclude deformities of the long bones of the legs and typicalcraniofacial changes (bossing of the skull, prominent malar emi-nence, depression of the bridge of the nose, tendency to a mon-goloid slant of the eye, and hypertrophy of the maxillae, whichtends to expose the upper teeth).1 Individuals who have not beenregularly transfused usually die before the third decade.

Beta-thalassemia intermediaPatients with thalassemia intermedia show a markedly het-

erogeneous clinical picture. Principle symptoms are pallor,jaundice, cholelithiasis, liver and spleen enlargement, moderateto severe skeletal changes, leg ulcers, extramedullary masses ofhyperplastic erythroid marrow, a tendency to develop osteope-nia and osteoporosis, and thrombotic complications resultingfrom a hypercoagulable state because of the lipid membranecomposition of the abnormal red blood cells (particularly insplenectomized patients).1,12 By definition, transfusions are notrequired or only occasionally required. Iron overload occursmainly from increased intestinal absorption of iron caused byineffective erythropoiesis.

Recently, the mechanism of iron hyperabsorption in beta-thalassemia has been at least partly elucidated. Iron absorptionis essentially controlled by hepcidin, a small peptide secreted bythe hepatocytes, which blocks iron uptake in the intestine andiron release from the reticuloendothelial system.13 Hepcidinbinds ferroportin, an iron transporter present on the surface ofabsorptive enterocytes, macrophages, and hepatocytes, and thehepcidin-ferroportin complex is internalized and rapidly de-graded, thereby explaining the defective intestinal iron absorp-tion.14 Transcription of hepcidin is controlled by bone morpho-genetic proteins (BMP), which activate the SMAD proteincomplex that translocates to the nucleus and stimulates hepcidintranscription.15 Hepcidin expression is enhanced by iron over-load and inflammation, whereas it is inhibited by anemia andhypoxia.

Recent studies have shown that serum from untransfusedthalassemia patients have high level of growth differentiationfactor 15, a member of the transforming growth factor super-family-like BMPs, whose production is related to the expansionof the erythroid compartment. Growth differentiation factor 15inhibits hepcidin expression by opposing the effect of BMP,thereby leading to intestinal iron hyperabsorption and increasediron release from macrophages.16 Consequently, the secretion of

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ferritin is reduced and its serum level relatively decreased.Therefore, in thalassemia intermedia, the determination of se-rum ferritin underestimates the extent of iron accumulation. Incontrast, in thalassemia major, red cell transfusions decrease theerythropoietic drive and increase iron overload, resulting inrelatively high hepcidin level, which reduces dietary iron ab-sorption and iron release from macrophages. This may contrib-ute to iron accumulation in Kupffer cells17–19 (Fig. 1). Theassociated complications of iron overload may present later, butit may be as severe as those seen in individuals with thalassemiamajor who depend on transfusions.

HEMATOLOGICAL FEATURES

Beta-thalassemia carrier stateCarriers of beta-thalassemia are clinically asymptomatic. The

characteristic hematological features are microcytosis (reducedred blood cell volume), hypochromia (reduced red blood cell Hbcontent), increased HbA2 level (the minor component of theadult Hb, which is made up of two alpha and two delta chains[alpha2delta2]), and slightly imbalanced alpha/beta � gammaglobin chain synthesis ratio (1.5–2.4), by in vitro synthesis ofradioactive-labeled globin chains.

The Hb pattern of beta-thalassemia heterozygotes is charac-terized by 92–95% HbA, �3.8 HbA2, and variable amount ofHbF (0.5–4%). Examination of the blood smear reveals micro-cytosis, hypochromia, and marked variations in size and shapeof the red blood cells.

Thalassemia majorPatients with thalassemia major have a severe microcytic and

hypochromic anemia, associated with increased number of redblood cells and low mean corpuscular volume (MCV) and meancorpuscular Hb (MCH). Peripheral blood smear shows, in ad-dition to microcytosis and hypochromia, anisocytosis, poikilo-cytosis (spiculated tear drop and elongated cells), and nucleatedred blood cells (i.e., erythroblasts). The number of erythroblastsis related to the degree of anemia and is markedly increasedafter splenectomy.

Hb pattern (by cellulose acetate electrophoresis or high-performance liquid chromatography [HPLC]) varies according

to the type of beta-thalassemia. In beta0-thalassemia, character-ized by the lack of beta globin chain synthesis, HbA is absent,HbF is 95–98%, and HbA2 is 2–5%. In beta�-thalassemiahomozygotes with a residual variable beta globin synthesis orbeta0/beta� compound heterozygotes, the Hb pattern showsHbA between 10 and 30%, HbF in the order of 70–90%, andHbA2 of 2–5%.

Bone marrow examination is usually not necessary for diag-nosis of affected individuals. Bone marrow is extremely cellu-lar, mainly as a result of marked erythroid hyperplasia, with amyeloid/erythroid ratio reversed from the normal 3 or 4 to 0.1or less.

In vitro synthesis of radioactive-labeled globin chains inaffected individuals reveals in beta0-thalassemia a completeabsence of globin beta chains and a marked excess of alphaglobin chains compared with gamma globin chains; the alpha/gamma ratio is �2.0. In beta�-thalassemia, the variable degreeof reduction of beta globin chains results in severe (thalassemiamajor) to mild (thalassemia intermedia) clinical phenotypes.The imbalance of the alpha/beta � gamma ratio is similar tothat in beta0-thalassemia major.

Thalassemia intermediaPatients with thalassemia intermedia have a moderate anemia

and show a markedly heterogeneous hematological picture,ranging in severity from that of the beta-thalassemia carrierstate to that of thalassemia major.

MOLECULAR GENETICS

Beta-thalassemiaThe beta globin (HBB) gene maps in the short arm of chro-

mosome 11 in a region also containing the delta globin gene, theembryonic epsilon gene, the fetal A-gamma and G-gammagenes, and a pseudogene (�B1). The five functional globingenes are arranged in the order of their developmental expres-sion (Fig. 2).

The HBB gene, which spans 1.6 Kb, contains three exons andboth 5� and 3� untranslated regions (UTRs). The HBB is regu-lated by an adjacent 5� promoter in which a TATA, CAAT, andduplicated CACCC boxes are located. A major regulatory re-

Fig. 1. Mechanism of control of hepcidin synthesis. Bone morphogenetic protein (BMP) through the BMP-HJVcoreceptor activates SMAD protein complex, which translocates to the nucleus, acts on the promoter, and activateshepcidin production.

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gion, containing also a strong enhancer, maps 50 Kb from thebeta globin gene (Fig. 2). This region, dubbed locus controlregion (LCR), contains four (HS-1 to HS-4) erythroid specificDNAse hypersensitive sites (HSs), which are a hallmark ofDNA-protein interaction. Each HS site is constituted by a com-bination of several DNA motifs interacting with transcriptionfactors, among which the most important are GATA-1 (GATAindicates the relative recognition motif), nuclear factor erythroid2, erythroid Kruppel-like factor, and friend of GATA 1.Theimportance of LCR for the control of the beta-like globin geneexpression has been discovered by studying a series of naturallyoccurring deletions that totally or partly remove the HS sitesand result in the inactivation of the intact downstream betaglobin gene. Several transcription factors bind and regulate thefunction of the HBB gene, the most important of which iserythroid Kruppel-like factor 1, which binds the proximalCACCC box, and whose knockout in the mouse leads to abeta-thalassemia-like clinical picture.

Beta-thalassemias are heterogeneous at the molecular level.More than 200 disease-causing mutations have been so faridentified. The majority of mutations are single nucleotide sub-stitutions or deletions or insertions of oligonucleotides leadingto frameshift. Rarely beta-thalassemias result from grossgene deletion. A complete updated list of beta-thalassemiamutations is available through the Globin Gene Server WebSite (http://www.globin.cse.psu.edu).

Point mutations affecting the beta globin expression belongto three different categories: mutations leading to defectivebeta-gene transcription (promoter and 5� UTR mutations); mu-tations affecting messenger RNA (mRNA) processing (splice-junction and consensus sequence mutations, polyadenylation,and other 3� UTR mutations); and mutations resulting in abnor-mal mRNA translation (nonsense, frameshift, and initiationcodon mutations).

Beta0-thalassemias, characterized by the complete absence ofbeta chain production result from deletion, initiation codon,nonsense, frameshift, and splicing mutations, especially at thesplice-site junction. On the other hand, beta�-thalassemias,characterized by reduced production of the beta chains, areproduced by mutations in the promoter area (either the CACCCor TATA box), the polyadenylation signal, and the 5� or 3� UTRor by splicing abnormalities. According to the extent of thereduction of the beta chain output, the beta�-thalassemia mu-tations may be divided into severe, mild, and silent. A list of themild and silent mutations is reported in Table 1.

The silent mutations, which are characterized by normalhematological findings and defined only by a mildly imbalancedalpha/beta globin chain synthesis ratio, result from mutations of

the distal CACCC box, the 5� UTR, the polyadenylation signaland some splicing defects. The mild mutations show moderatethalassemia-like hematological features and imbalanced globinchain synthesis and are produced by mutations in the proximalCACCC box, TATA box, 5� UTR, or exon 1, causing alterna-tive splicing (Hb Malay, HbE, and codon 24 T3A), or in the

Table 1 Mild and silent HBB gene mutations causingbeta-thalassemia

Mutation type or location Mild beta� Silent

Transcriptional mutantsin the proximalCACC box

�90 C�T �101 C�T

�88 C�T �92 C�T

�88 C�A

�87 C�T

�87 C�G

�87 C�A

�86 C�T

�86 C�G

TATA box �31 A�G

�30 T�A

�29 A�G

5� UTR �22 G�A

�10-T

�33 C�G �1� A�C

Alternative splicing cd19 A�C(Hb Malay)

cd27 G�T(Hb Knossos)

cd24 T�A

Consensus splicing IVS1-6 T�C

Intron IVS2-844 C�G

3� UTR �6 C�G

Poly-A site AACAAAAATGAA

AATAAG

Mild beta0—frameshift cd6-AA

cd8-AA

Fig. 2. Chromosome localization and structure of alpha and beta globin gene clusters (for details see text).

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consensus splicing sequence, 3� UTR, and poly-A site. Muta-tions activating a cryptic splicing site in exon 1, at codons 19,26, and 27, are associated with a mild or silent phenotype,because of the preferential use of the normal splice site, andresult in the production of the abnormal Hb Malay, HbE(extremely common in South East Asia), and Hb Knossos,respectively.

A few beta0-thalassemias display a mild phenotype (forinstance, cd6-A and cd 8-AA) because of the linkage with thenondeletion promoter mutation of the G-gamma gene (�158G-gamma), which is associated with high production of HbFduring hematologic stress.

Deletions affecting the beta globin gene are very rare, exceptfor a 619-bp deletion removing the 3� end of the beta globingene, which is relatively common in Sind and Punjab popula-tions of India and Pakistan. Another group of deletions (com-plex beta-thalassemia), in addition to the beta globin gene,involve also the delta (delta-beta0-thalassemia), the delta andA-gamma genes (G-gammaA-gammadeltabeta0-thalassemia),or the whole beta globin gene cluster. Finally, partial or totaldeletions of the LCR, but leaving the beta globin gene intact,inactivate the beta globin gene.

Despite the marked molecular heterogeneity, the prevalentmolecular defects are limited in each at risk population (Fig. 3),

in which 4–10 mutations usually account for most of the betaglobin disease-causing allele.

Beta-thalassemic hemoglobinopathiesUnder this category are included different structurally abnor-

mal Hbs associated with a beta-thalassemia phenotype. Threedifferent molecular mechanisms are responsible for these phe-notypes: activation of cryptic splice site in exons, leading, forinstance, to the production of HbE (already mentioned); forma-tion of a delta-beta hybrid gene; or mutation causing hyperun-stable beta globin.

The delta-beta hybrid genes leading to beta-thalassemia aredubbed Hb Lepore genes and are made up of N-terminal aminoacid sequence of the normal delta chain and the C terminalsequence of the normal beta chain. Depending on the codon oftransition from delta to beta sequences, different Hb Leporegenes have been described. In peripheral blood, Hbs Lepore arepresent in low percentage of total Hb (around 10% in carriers)because of the low production, which depends on the reducedactivity of the delta promoter and on the relative instability ofthe variant Hb. These characteristics explain why Hbs Leporeare considered a beta-thalassemia-like mutation.

Hyperunstable beta globins comprehend a group of betaglobin mutants, which result in the production of Hb variants

Fig. 3. Most common beta-thalassemia mutations in different countries.

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that are extremely unstable and precipitate before assemblingwith the alpha chains to produce the Hb tetramer. This results inineffective erythropoiesis, which is exacerbated by the concom-itant relative excess of the alpha chains, thereby leading tophenotypic thalassemia-like manifestations (more frequently inthalassemia intermedia than in the heterozygous state).

Beta-thalassemia-like mutations mapping outside thebeta globin gene cluster

In rare instances, the beta-thalassemia defect does not lie inthe beta globin gene cluster. Some mutations in the X-linkedtranscription factor GATA-1 may produce thrombocytopeniaassociated with thalassemia trait, whereas molecular lesionsaffecting the general transcription factor TFIIH result, in addi-tion to thalassemia-like hematological features, in xerodermapigmentosum and trichothiodystrophy.20,21

Molecular diagnosis of beta-thalassemiaCommonly occurring mutations of the HBB gene are de-

tected by a number of polymerase chain reaction (PCR)-basedprocedures. The most commonly used methods are reverse dotblot analysis or primer-specific amplification with a set ofprobes or primers complementary to the most common muta-tions in the population from which the affected individualoriginated.22 Other methods based on real-time PCR or microar-ray technology because of their reproducibility, rapidity, andeasy handling are potentially suitable for the routine clinicallaboratory.23,24

If targeted mutation analysis fails to detect the mutation,scanning or sequence analysis can be used. Sensitivity of bothmutation scanning and sequence analysis is 99%. In the mean-time, the presence of an extended deletion should be investi-gated by using multiplex ligation-dependent probe amplification(MPLA).

PHENOTYPE-GENOTYPE CORRELATION

Homozygous beta-thalassemiaHomozygosity or compound heterozygosity for beta-thalas-

semia most commonly result in the clinical phenotype of trans-fusion-dependent thalassemia major. However, a consistent pro-portion of homozygotes develop milder forms, calledthalassemia intermedia, which range in severity from thalasse-mia major to the beta-thalassemia carrier state.25–29

To understand the clinical-molecular relationships, weshould remember that the main pathophysiological determinantof the severity of the beta-thalassemia syndromes is the extentof alpha/nonalpha globin chain imbalance (see Introduction).Therefore, any factor capable of reducing the alpha/nonalphachain imbalance may have an ameliorating effect on the clinicalpicture.

The most clinically important mechanism consistently result-ing in thalassemia intermedia is the coinheritance of homozy-gosity or compound heterozygosity for mild beta-thalassemiaalleles, namely a beta-thalassemia defect associated with aconsistent residual output of beta chains from the affected betaglobin locus (see Molecular genetics). The most common mildmutations are beta�IVS-I nt 6 (T3C)—found in the Mediter-ranean area, and beta codon 26 (G3A), which gives rise toHbE, prevailing in South-East Asia (Table 1). By contrast,compound heterozygotes for a mild and a severe defect result ina spectrum of phenotypes ranging from severe to mild forms.Therefore, from the clinical point of view, in presence of a mild

beta-thalassemia/severe beta-thalassemia genotype, we cannotpredict the development of mild clinical picture.

The mildest beta-thalassemia alleles are the silent alleles(Table 1), which show normal hematological features and canbe identified solely by a slight imbalance of alpha/nonalphaglobin chain synthesis ratio.30–32 Homozygosity for silent al-leles produces a very mild form of thalassemia intermedia. Mildbeta-thalassemia also results from the beta-silent/beta-mild orbeta-silent/beta-severe genotypes.

It should be pointed out that, as we will see later on, even thehomozygous beta0 or severe beta�-thalassemia may develop anattenuated form resulting from coinherited modifying amelio-rating genetic factors.

The second mechanism leading to mild beta-thalassemia isthe coinheritance of homozygous beta-thalassemia and an al-pha-thalassemia determinant that, by reducing the alpha chainoutput, decreases the alpha/nonalpha chain imbalance. A singlealpha globin gene deletion is sufficient to improve the clinicalphenotype of homozygous beta�-thalassemia, whereas in beta0-thalassemia, the deletion of two alpha globin genes or thepresence of an inactivating mutation of the major alpha2 globingene is necessary.26,33,34

The third mechanism is the coinheritance of a genetic deter-minant that is able to sustain a continuous production of gammachains in adult life, thereby reducing the extent of the alpha/nonalpha chain imbalance. The nature of the beta-thalassemiamutation per se may affect the ability to produce gamma chains.This mechanism occurs in delta beta0-thalassemia, which isbecause of deletions of variable extent within the beta globincluster, and in those more limited deletions involving only the5� region of the beta globin promoter. In other cases, the reasonfor the high gamma chain output depends on the co-transmis-sion of a nondeletion form of hereditary persistence of HbF(HPFH), caused by point mutations at G-gamma or A-gammapromoters (�196 C3T A-gamma; �158 C3T G-gamma). AC3T mutation at position �196 A-gamma has been found tobe associated in cis with the codon 39 nonsense mutation insome Sardinian beta-thalassemia chromosomes (Sardiniandeltabeta0-thalassemia).35 The increase in gamma chain produc-tion from the �196 A-gamma gene probably compensates forthe absence of beta chain production from the affected betalocus. Compound heterozygosity for this determinant and fortypical beta–thalassemia, thus, develops thalassemia interme-dia. Homozygotes for the Sardinian deltabeta0-thalassemia areclinically normal and can be detected solely by hematologicalanalysis.36 Heterozygotes for Sardinian deltabeta-thalassemiahave thalassemia-like hematological features and high HbFlevels.37 In contrast, C3T �158 G-gamma is silent both innormal subjects and beta-thalassemia heterozygotes but leads toa high HbF production rate during hematopoietic stress, asoccurs in homozygous beta-thalassemia or sickle cell anemia.38

The �158 G-gamma mutation may be associated with IVS II nt1 (G3A), frameshift 8 (AA), frameshift 6 (-A), and someonewith codon 39 nonsense mutations, thereby explaining the mildphenotype possibly associated with these mutations.

Coinheritance of genetic determinants capable of sustaining acontinuous production of HbF in adult life and mapping outsidethe beta globin cluster may also determine a mild phenotype. Itshould be noted that the residual amount of HbF in normaladults is unevenly distributed and mainly contained in a sub-population of red blood cells, denominated “F cells.” Both HbFand F-cells percentage, in normal adults and in beta-thalassemiaheterozygotes, show a wide variation quantitative trait loci(QTLs)with a continuous distribution.38 Those individuals witha moderate increase of HbF in the order of 0.8–5% , with an

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heterocellular distribution, are indicated as carriers of hetero-cellular HPFH. So far, two HPFH have been mapped on chro-mosome 2p16 and chromosome 6q23.39–41The locus on chro-mosome 2p16 has been located by genome-wide associationstudy on the BCL11A gene, and the single nucleotide polymor-phism (SNP) rs11886868 in its intron 2 was found stronglyassociated with HbF levels. Furthermore, the C allele of thisSNP was significantly higher in Sardinian individuals withelevated HbF levels (HPFH). The BCL11A variants were shownto influence HbF levels also in nonanemic Caucasians from aEuropean twin study.40 The same C variant was significantlyhigher in beta0-thalassemia homozygotes for the codon 39 non-sense mutation with a mild phenotype (thalassemia intermedia),as compared with those with the same beta globin genotype, butwith a severe phenotype, compensating for the imbalance of Hbproduction through the augmentation of HbF levels.39 Further-more, the BCL11A variant C allele by increasing HbF levels wasassociated with a mild phenotype also in sickle cell disease.39,42

In conclusion, the BCL11A gene is able to modify the phenotypeof homozygous beta-thalassemia and sickle cell anemia byincreasing HbF levels. The BCL11A gene encodes a zinc-fingertranscription factor, which regulates the globin switching duringontogeny by interacting with specific sequence in the betaglobin cluster and repressing the HbF expression43,44

According to these results, the identification of BCL11Apolymorphism in young homozygous beta-thalassemia andsickle cell anemia patients may serve as a prognostic indicationfor the severity of the disease. Furthermore, targeted down-regulation of BCL11A in patients could elevate HbF levels,thereby ameliorating the severity of these inherited anemias.

Another HPFH locus has been mapped many years ago onchromosome 6q23 by linkage analysis in a large Indian familywith segregating beta-thalassemia45 and confirmed more re-cently by twin studies.38 Further studies have shown that thechromosome 6 genetic variant responsible for HbF variationmaps in the HBS1L-MYB region. Specifically, the G allele ofSNP rs9389268 is associated with high HbF levels. The causalvariant in this region and its mechanism for increasing HbF isnot yet known. HBS1L is a putative member of the “GTPasesuperfamily,” whereas MYB has a crucial role in normal eryth-ropoiesis. Recent studies have shown that the HBS1L-MYBintergenic polymorphisms contain regulatory sequences con-trolling MYB expression.46 However, further studies are neces-sary to elucidate the biological role of these two genes in themodulation of HbF. The HBS1L-MYB locus contributes 3–7%of trait variance,39,42 whereas the BCL11A variant explain7–12%.42,47

Two QTL loci mapping on chromosome 848,49 and chromo-some X,50,51 respectively, have not been validated in subsequentgenome-wide linkage and association studies.39,40

The loci-modulating HbF levels explain only partially itsvariation. This consideration clearly indicates the existence ofother QTL traits for HbF in the human genome. Recent studiesfrom our group indicate that the BCL11A, HBS1L-MYB region,and alpha-thalassemia contribute differently in the ameliorationof clinical severity in thalassemia intermedia (odd ratio 5.15,4.61, and 3.3, respectively). Taken together, the loci are able tocorrectly predict 75% of the phenotypes of homozygous beta0-thalassemia.52

In conclusion, despite the significant progress made in thisfield of thalassemia disorders, in a significant number of casesof thalassemia intermedia, the molecular mechanism has notbeen so far elucidated.

Heterozygous beta-thalassemiaFrom the clinical point of view, heterozygous beta-thalasse-

mia, whether beta0 or beta�, is completely asymptomatic and ischaracterized hematologically by high red blood cell count,microcytosis, hypochromia, increased HbA2 levels, and unbal-anced alpha/nonalpha globin chain synthesis. However, severalenvironmental or genetic factors may modify this phenotype,leading either to thalassemia intermedia, despite the presence ofa single beta globin gene affected, or to hematologically atyp-ical carrier states (see Beta-thalassemia carrier identification).

To date, two mechanisms have been identified, which mayincrease the clinical and hematological severity of beta-thalas-semia heterozygotes. The first is related to the coinheritance ofboth heterozygous beta-thalassemia and triple or quadruple al-pha globin gene arrangement, which, by increasing the magni-tude of imbalance of alpha/non alpha globin chain synthesis,may cause an excess of unassembled alpha chains, therebyresulting in premature destruction of red blood cell precursors.Beta-thalassemia carriers who are heterozygous or homozygousfor the triplicated alpha globin gene arrangement may indeeddevelop a clinical phenotype of intermediate severity.53–56 Sim-ilarly, phenotype of thalassemia intermedia has also been seenin beta-thalassemia carriers who have inherited a chromosomecontaining the quadruplicated alpha globin gene arrange-ment.57–59 However, normal people carrying the triplicated orquadruplicated alpha globin gene arrangement have a normalphenotype most likely, because the small excess of alpha chainsthat is synthesized can be eliminated by proteolysis.

The other mechanism increasing the severity in beta-thalas-semia heterozygotes depends on the presence of a mutation inthe beta globin gene, which causes an extreme instability of thebeta globin chains.60,61 Heterozygotes for this condition aremore severely affected than subjects with the beta-thalassemiacarrier state, because in addition to producing an excess of alphachains, they synthesize highly unstable beta chains that bindheme and precipitate in red blood cell precursors before assem-bling with the alpha chains. This leads to the production ofinclusion bodies made up of alpha and unstable beta chains.Beta-thalassemia resulting from hyperunstable beta chains istransmitted in a dominant fashion or may result from a de novomutation.

Mutations leading to hyperunstable beta chains include mis-sense mutations, minor deletions leading to loss of intact codon,and frameshifts. Most mutations in the phase terminationcodons that result in dominant beta-thalassemia lie in exon 3,whereas those producing the typical recessively inherited formsare located in exons 1 or 2. In exons 1 or 2 mutations, very littlebeta globin mRNA is found in the cytoplasm of red blood cellprecursors, whereas exon 3 mutations are associated with asubstantial amount of abnormal cytoplasmic mRNA. This leadsto the synthesis of truncated beta chains products, which areunstable and thereby act in a dominant-negative fashion, caus-ing premature destruction of red blood cells. This differencedepends on the fact that premature termination codon mutationslying in exons 1 or 2 activate the process of nonsense-mediatedmRNA decay, therefore precluding the accumulation of mRNAencoding for truncated peptides.62 The diagnosis of dominantbeta-thalassemia is difficult, because the unstable beta globinchains in peripheral blood are not easily detected, not even byvery accurate electrophoretic or chromatographic procedures.The suspicion for such conditions should derive from the pres-ence of beta-thalassemia-like disorders of intermediate severityarising de novo or transmitted according to a dominant pattern.

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Exceptionally, beta-thalassemia intermedia may develop insubjects heterozygous for beta-thalassemia because of a mosaicsomatic deletion of the in trans beta globin gene in a subpopu-lation of hematopoietic cells.63,64

Compound heterozygosity for beta-thalassemia and somebeta chain structural variant (HbD-Los Angeles beta 121Glu3Gln; HbC beta 6 Glu3Lys; HbO-Arab beta 121Glu3Lys) may produce thalassemia intermedia as a result ofglobin chain imbalance in combination with the modified struc-tural and functional characteristic of the variants.65

Finally, the proposed role, if any, of the alpha-Hb stabilizingprotein (AHSP) as a modulating factor of the phenotype has notyet clarified.66 AHSP forms a stable complex with free al-pha-Hb and protect free alpha chain from precipitation, therebyacting as a specific alpha-Hb molecular chaperone. In the mousesystem, knockout of AHSP leads to reduced lifespan of circu-lating red blood cells, causing increased apoptosis of erythroidprecursors and exacerbating the severity of heterozygous beta-thalassemia, which usually displays a thalassemia intermediaphenotype.66 The studies of AHSP in humans led to inconstantresults. It seems, however, that variation of the AHSP level maybe able to aggravate the phenotype of simple heterozygotesfor beta-thalassemia.67,68 The most common ascertainedmechanism leading to thalassemia intermedia are summa-rized in Table 2.

Other clinical genetic modifiersIn addition to the variation of the phenotype resulting from

allelic heterogeneity at the beta globin locus, from the effect ofalpha or gamma globin gene mutation or from coinheritance ofheterocellular HPFH delineated above, the phenotype of beta-thalassemia could also be modified by the action of geneticfactors mapping outside the globin gene cluster and notinfluencing the HbF. Among these factors, the ones bestdelineated so far are those affecting bilirubin, iron, and bonemetabolisms.69

Because of the rapid turnover of red cell precursors and theresulting breakdown of the heme products, both homozygotesand heterozygotes for beta-thalassemia may develop mild jaun-dice and have the propensity to gallstone formation. In beta-

thalassemia, the level of indirect bilirubin and gallstone forma-tion are related to a polymorphic motif in the promoter of thegene involved in the hepatic glucuronidation of bilirubin,namely bilirubin UDP-glucuronosyltransferase (UGT1A). Innormal individuals, the promoter has six TA repeats in theTATA box (TA)6. Homozygotes for an additional repeat (TA)7

develop mild unconjugated hyperbilirubinemia (Gilbert syn-drome) because of less efficient activity of UGT1. Studiesperformed in the last few years in thalassemia major, thalassemiaintermedia, and beta-thalassemia carrier state have shown thatpatients developing hyperbilirubinemia, jaundice, and gallstonesusually have the more common and less efficient TA7 motif.70–75

It should be pointed out that the same phenomenon has beenobserved in many varieties of hemolytic anemia.76–79 These find-ings have led us to conclude that the Gilbert syndrome mutationacts as a modifying gene in beta-thalassemia by determining orpromoting the development of jaundice or gallstones.

Patients with beta-thalassemia, especially when not well che-lated, develop extensive iron accumulation in many tissuesincluding liver, heart, and endocrine glands, in part, because ofthe destruction of transfused red blood cells and, in part, par-ticularly in thalassemia intermedia, because of increased ironabsorption. Some studies seem to indicate that the commonmutation of the HFE gene (C282Y), which causes the commontype of HH, might be involved in determining the variability ofiron overload in patients with thalassemia intermedia.80 Longoet al.81 by studying a large group of thalassemia major patientsfound that the presence of a single mutation in HFE gene(C282Y and H63D) does not influence the severity of ironloading, assessed by serum ferritin and liver iron concentration,likely because the effect of the mutations on iron overload ishidden as a result of treatment (i.e., posttransfusional ironoverload and iron chelation). Furthermore, homozygosity forthe H63D mutation, whose functional significance in HH is stillbeing evaluated, when coinherited with heterozygous beta-thalassemia seems to determine an increase in iron overload.82

Conversely, coinherited heterozygosity for beta-thalassemiaseems to increase the rate of iron accumulation in C282Yhomozygotes.83

Another common complication, in adults with beta-thalasse-mia, is the development of marked and progressive osteoporo-sis, which depends on many factors including hypogonadismand extent of iron chelation. However, recent evidence seems toindicate that the development of this complication may be alsorelated to polymorphisms of the genetic loci involved in bonemetabolism, namely vitamin D receptor and the COLIAIgene.84–88

Finally, it should be pointed out that there is marked evidenceindicating that the high frequency of beta-thalassemia in certainareas of the world is related to heterozygote advantage vis-a-visPlasmodium falciparum malaria.89 Exposure to malaria, how-ever, resulted also in the expansion of polymorphisms at manyother genetic loci, including human leukocyte antigen (HLA),tumor necrosis factor alpha, and intercellular adhesion mole-cule-1, which have an important role in the defense mechanismsagainst infectious diseases.90–92 This consideration may indi-cate that children with beta-thalassemia may respond to infec-tions differently than normal children.

BETA-THALASSEMIA CARRIER IDENTIFICATION

The typical phenotype of the beta-thalassemia trait, essen-tially characterized by reduced MCV, MCH, and increasedHbA2, may be modified by several coinherited genetic factor,which may cause problems in carrier identification (Table 3).

Table 2 Ascertained molecular mechanisms leading tothalassemia intermedia

Homozygotes or compound heterozygotes

● Beta-thalassemia mutations

a. Mild mutation

b. Silent mutation

c. Mild/silent mutation

● Coinherited alpha-thalassemia

a. Single alpha-globin gene deletion (�alpha/alpha alpha)

b. Deletion of two alpha-globin gene (�alpha/�alpha or��/alpha alpha)

c. Point mutations of the major alpha 2 globin gene

● Genetic determinant of high HbF production

a. due to the beta-thalassemia mutation per se(deltabeta thalassemia, beta promoter deletion)

b. coinherited Agamma or Ggamma promoter mutation (�158Ggamma (A¡T; �196 Agamma (C¡T)

c. heterocellular HPFH, BCL11A on chromosome 2 andHBS1L-MYB region on chromosome 6

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Coinheritance of heterozygous beta-thalassemia and alpha-thalassemia may raise the MCV and the MCH, high enough todetermine normal values at least in some of these double het-erozygotes. This may occur as a result of either a deletion of twoalpha globin structural genes or as a nondeletion lesion affectingthe major alpha globin gene (the two functional alpha genes,denominated as alphal and alpha2, have a relative expression of1:3). Fortunately, these carriers may be easily identified for theirhigh HbA2 levels.93,94

Elevation of HbA2 is the most important feature in thedetection of heterozygous beta-thalassemia, but a substantialgroup of beta-thalassemia heterozygotes may have normalHbA2. The first mechanism to account for the abnormally lowHbA2 levels in a beta-thalassemia carrier is the presence of aspecific mild beta-thalassemia mutation, such as the beta�

IVS-I nt 6 mutation.95 A second common mechanism is thecoinheritance of heterozygous beta-thalassemia and delta-thalassemia. The decreased output of the delta globin chainsmay result in normalization of HbA2 levels.96,97 Also, gammad-eltabeta- and deltabeta-thalassemia carriers have normal HbA2.However, all these normal-HbA2 atypical heterozygotes havelow MCV and MCH. Because of this phenotype, normal HbA2

beta-thalassemia heterozygotes should be differentiated fromalpha-thalassemia heterozygotes by globin chain synthesis anal-ysis and/or by alpha, beta, and delta globin gene analysis.Deltabeta-thalassemia, in addition, may easily be defined by thevariable but markedly increased HbF.

Another major problem in carrier screening is the identifica-tion of silent beta-thalassemia or the triple or quadruple alphaglobin gene arrangement, both of which may lead to the pro-duction of intermediate forms of beta-thalassemia by interactingwith typical heterozygous beta-thalassemia. Silent beta-thalas-semias are characterized by normal MCV and MCH values andnormal HbA2 and by the fact that they are defined only by theslight imbalance in the alpha/nonalpha globin synthesis.

Nevertheless, on examining the hematological features ofthese carriers, one may find borderline HbA2 or MCV and MCHvalues, which may alert for the presence of atypical beta-thalassemia, thus requiring further studies (globin chain synthe-sis or gene analysis). The most common silent beta-thalassemiais the beta� �101 C3T mutation; others are very rare.98 Thetriple-quadruple alpha globin gene arrangement may show aslight imbalance of alpha/nonalpha chain synthesis or, morecommonly, may be completely silent. An extreme, althoughrare, instance of thalassemia gene combination, which may

result in carrier identification, is the coinheritance of alpha,delta, and beta-thalassemia, which may lead to a completelysilent phenotype.99

Carrier detection procedureSeveral procedures have been proposed for beta-thalassemia

carrier screening.100 The cheapest and simplest is based onMCV and MCH determination, followed by HbA2 quantitationfor subjects showing microcytosis (low MCV) and reduced Hbcontent per red blood cell (low MCH). However, because withthis procedure a considerable proportion of double heterozy-gotes for beta- and alpha-thalassemia may be missed (these arefound in many populations, such as Sardinians, where bothdisorders are common), it can only be used in populations witha low frequency of alpha-thalassemia. At our center in Cagliari,in the first set of examinations, we include MCV and MCHdetermination and Hb chromatography by HPLC, which canquantitate HbA2 and HbF and can detect the most common Hbvariants (HbS, HbC, and HbE) that may result in a Hb disorderby interacting with beta-thalassemia (Fig. 4). It should be statedthat HPLC is also capable of detecting Hb Knossos, a mildbeta-thalassemia allele, which is not identified by using com-mon procedures for Hb analysis. In the presence of low MCVand MCH and elevated HbA2 levels, a diagnosis of heterozy-gous beta-thalassemia is made. A phenotype characterized bymicrocytosis, hypochromia, normal-borderline HbA2, and nor-mal HbF may result from iron deficiency, alpha-thalassemia,gammadeltabeta-thalassemia, beta � delta-thalassemia, or mildbeta-thalassemia. After excluding iron deficiency through ap-propriate studies (red blood cell, zinc protoporphyrin determi-nation, and transferrin saturation), the different thalassemiadeterminants leading to this phenotype are discriminated byglobin chain synthesis analysis and eventually by alpha, beta,and delta globin gene analysis.100 In the presence of normalMCV and borderline HbA2 levels, we are inclined to suspect thepresence of a silent mutation or the triple or quadruple alphaglobin gene arrangement and, therefore, proceed directly toalpha- and beta globin gene analysis, because the alpha/betaglobin chain synthesis ratio could also be normal.101 Definitionof the type of thalassemias in these carriers is solely recom-mended when they mate with a carrier of a typically high HbA2

beta-thalassemia or an undetermined type of thalassemia. Inthose rare cases showing normal or low MCV-MCH, normal orreduced HbA2 levels, and high HbF, we suspect the presence ofdeltabeta-thalassemia, which should be differentiated fromHPFH. This distinction is performed by globin chain synthesisanalysis (normal in HPFH and unbalanced in deltabeta-thalas-semia) or beta-cluster gene analysis or both.

Molecular diagnosis of modifying genesMolecular diagnosis is carried out in patients affected by

homozygous beta-thalassemia for defining the genotype, whichmay be useful for predicting the severity of the disease, and incarriers identified by hematological analysis.

The procedures available to detect the beta-thalassemia mu-tation have been already described.23,24As previously men-tioned, delta globin gene analysis may be necessary to definedouble heterozygotes for delta- and beta-thalassemia that maybe mistaken for alpha-thalassemia trait. The suspicion of inter-acting delta-thalassemia may arise when borderline HbA2 levelsare found or when family studies show segregating delta-thalas-semia (characterized by normal MCV-MCH and low HbA2) and

Table 3 Heterozygous beta-thalassemia: phenotypemodification

Phenotype Genotype

Normal cell indices - Alpha and beta thalassemia interaction

Normal HbA2 level - Iron deficiency- Coinheritance of delta and

beta-thalassemia- Some mild beta-thalassemia mutations- Gamma delta beta-thalassemia

Normal red cell indicesand HbA2 level (silent)

- Silent beta-thalassemia mutations- Alpha-globin gene triplication/

quadruplication

Severe heterozygousbeta-thalassemia

- Hyperunstable hemoglobin- Coinheritance of heterozygous

beta-thalassemia and triplicatedor quadruplicated alpha-globin gene

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beta-thalassemia. However, identification of delta and betadouble heterozygotes may be accomplished by globin chainsynthesis analysis and/or alpha, beta, and delta globin geneanalysis.101,102

Definition of the delta-thalassemia mutation may be carriedout using one of the previously mentioned PCR-based methods.As in beta-thalassemia, also in delta-thalassemia, each popula-tion at risk has its own spectrum of common delta-thalassemiamutations that may be defined through a limited number ofspecific primers/probes. In Sardinians, for instance, few delta-thalassemia mutations have been so far detected. The list ofdelta-thalassemia mutations is available at the repository of thehuman beta and delta globin gene mutation. Although most ofthe delta-thalassemia determinants are in trans (on oppositechromosomes) to beta-thalassemia, some have also been de-tected in cis (on the same chromosome).103

Definition of the alpha globin gene arrangement may beperformed to discriminate between heterozygosity for alpha-thalassemia and double heterozygosity for delta- and beta-thalassemia or gammadeltabeta-thalassemia. This analysiscould also be useful in defining coinherited alpha-thalassemia inhomozygous beta-thalassemia, which may lead to the predictionof a mild clinical condition. Deletion alpha0- or alpha�-thalas-semias are detected by PCR using two primers flanking thedeletion breakpoint, which amplify a DNA segment only inpresence of specific deletions.104 Nondeletion alpha-thalassemiamay be detected by restriction endonuclease analysis or allelicoligonucleotide specific probes on selectively amplified alpha1

and alpha2 globin genes. Alpha globin gene triplication andquadruplication may be detected by the MPLA procedure.

Definition of coinherited HPFH determinants can be useful inpredicting the severity of the phenotype of an affected fetus. Asmentioned above, in fact, on increasing the gamma chain out-put, coinherited HPFH with homozygous beta-thalassemia maylead to a milder phenotype. The presence of high HbF in theparents may lead to the suspicion of double heterozygosity forbeta-thalassemia and HPFH. The 196 C3T in the A-gammagene and �158 C3T in the G-gamma gene mutations havebeen proved to be capable of ameliorating the clinical pheno-type of homozygous beta-thalassemia. The HPFH determinantsmay easily be detected through restriction endonuclease or dotblot analysis with oligonucleotide-specific probes on PCR-am-plified DNA. Furthermore, definition of the polymorphisms atthe BCL11A and HBS1L-MYB region may lead to predict thedevelopment of a specific mild phenotype.

If targeted mutation analysis fails to detect the mutation,mutation scanning or sequence analysis can be used to detectmutations in the HBB coding region (mutations in the noncod-ing region would not be detected by this analysis). Sensitivity ofboth mutation scanning and sequence analysis is 99%.

Deletions of variable extent of the beta gene or of the HBBcluster that result in beta-thalassemia or in the complex beta-thalassemias, called gammadeltabeta-thalassemia and deltabeta-thalassemia, are rare causes of beta-thalassemia and testing thatdeletions is available clinically by using MPLA.

Fig. 4. Flowchart for thalassemia carrier identification.

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POPULATION SCREENING

Because of the high carrier rate for HBB mutations in certainpopulations and the availability of genetic counseling and pre-natal diagnosis, population screening is ongoing in severalat-risk populations in the Mediterranean.105 Carrier testing re-lies on hematological analysis. When the hematological analysisindicates a beta-thalassemia carrier state, molecular genetictesting of HBB can be performed to identify a disease-causingmutation. If both partners of a couple have the HBB disease-causing mutation, each of their offspring has a 1⁄4 risk of beingaffected. Through genetic counseling and the option of prenataltesting, such a couple can opt to bring to term only thosepregnancies in which the fetus is unaffected.

The optimal time for the assessment of genetic risk, defini-tion of carrier status, and genetic counseling is before preg-nancy. It is appropriate to offer genetic counseling (includingdiscussion on the availability of prenatal diagnosis, potentialrisks to offspring, and reproductive options) to young adultswho are carriers.

Population screening associated with genetic counseling isextremely useful by allowing couples at risk to make informeddecision on their reproductive choices. Furthermore, in thepopulation at risk targeted by screening, a consistent reductionof the birth rate of affected children was registered, as shown inthe Sardinian population.

Prenatal diagnosisIn high-risk pregnancies in which both members are defined

carriers for beta-thalassemia, prenatal diagnosis is possible byanalysis of DNA extracted from fetal cells obtained by amnio-centesis, usually performed at approximately 15–18 weeks’gestation, or chorionic villus sampling at approximately 10–12weeks’ gestation. Both disease-causing alleles must be identi-fied before prenatal testing can be performed.

Alternatively, following an accurate genetic counseling ex-plaining the pros and cons of the procedure preimplantationgenetic diagnosis may be considered.

Prenatal diagnosis by analysis of fetal cells in maternal bloodis not yet available but is being investigated on a researchbasis.106,107 Finally, analysis of fetal DNA in maternal plasmafor the presence of the father’s mutation may lead to prenatalexclusion of homozygous beta-thalassemia. This testing is notyet clinically available but under investigation on a researchbasis with promising results.108,109

In indeterminate-risk pregnancies, either one parent is adefinite heterozygote and the other parent has a beta-thalasse-mia-like hematologic picture, but no HBB mutation has beenidentified by sequence analysis, or a mother is a known hetero-zygote and the father is unknown or unavailable for testing,especially if the father belongs to a population at risk. In eitherinstance, the options for prenatal testing should be discussed inthe context of formal genetic counseling. In indeterminate-riskpregnancies, the prenatal testing strategy is the analysis for theknown HBB mutation. If the known HBB mutation is present,analysis of globin chain synthesis is performed on a fetal bloodsample obtained by percutaneous umbilical blood sampling atapproximately 18–21 weeks’ gestation.

CLINICAL MANAGEMENT

A comprehensive review of the management of thalassemiamajor and thalassemia intermedia has been published byThalassemia International Federation and is available at theThalassemia International Federation Web site.110

Transfusion programIn thalassemia major, regular transfusions correct the anemia,

suppress erythropoiesis, and inhibit increased gastrointestinalabsorption of iron. Before starting the transfusions, it is abso-lutely necessary to carry out hepatitis B vaccination and per-form extensive red blood cell antigen typing, including Rh,Kell, Kidd, and Duffy and serum immunoglobulin determina-tion, the latter of which detects individuals with IgA deficiencywho need special (repeatedly washed) blood unit preparationbefore each transfusion. The transfusion regimen is designed toobtain a pretransfusion Hb concentration of 95–100 g/L. Trans-fusions are usually given every 2–3 weeks.

Treatment of individuals with thalassemia intermedia issymptomatic and based on splenectomy and folic acid supple-mentation. Treatment of extramedullary erythropoietic massesis based on radiotherapy, transfusions, or, in selected cases,hydroxyurea (with a protocol similar to that used for sickle celldisease). Hydroxyurea also increases globin gamma chains andmay have other undefined mechanisms. Because individualswith thalassemia intermedia may develop iron overload fromincreased gastrointestinal absorption of iron or from occasionaltransfusions, chelation therapy is started when the serum ferritinconcentration exceeds 300 �g/L.17,111

Transfusional iron overloadThe most common secondary complications are those related

to transfusional iron overload, which can be prevented by ade-quate iron chelation. After 10–12 transfusions, chelation ther-apy is initiated with desferrioxamine B (DFO), administered5–7 days a week by 12-hour continuous subcutaneous infusionvia a portable pump. Recommended dosage depends on theindividual’s age and the serum ferritin concentration. Youngchildren start with 20–30 mg/kg/day, increasing up to 40 mg/kg/day after the age of 5–6 years. The maximum dose is 50mg/kg/day after growth is completed. The dose may be reducedif serum ferritin concentration is low. By maintaining the totalbody iron stores below critical values (i.e., hepatic iron concen-tration �7.0 mg per gram of dry weight liver tissue), DFOtherapy prevents the secondary effects of iron overload, result-ing in a consistent decrease in morbidity and mortality.11 Ascor-bate repletion (daily dose not to exceed 100–150 mg) increasesthe amount of iron removed after DFO administration. Sideeffects of DFO are more common in the presence of relativelylow iron burden and include ocular and auditory toxicity,growth retardation, and, rarely, renal impairment and interstitialpneumonitis. DFO administration also increases susceptibilityto Yersinia infections. The major drawback of DFO chelationtherapy is low compliance resulting from complications ofadministration.

In clinical practice, the effectiveness of DFO chelation therapyis monitored by routine determination of serum ferritin concentra-tion. However, serum ferritin concentration is not always reliablefor evaluating iron burden, because it is influenced by other factors,the most important being the extent of liver damage.

Determination of liver iron concentration in a liver biopsyspecimen shows a high correlation with total body iron accu-mulation and is the gold standard for evaluation of iron over-load. However, (1) liver biopsy is an invasive technique involv-ing the possibility (though low) of complications; (2) liver ironcontent can be affected by hepatic fibrosis, which commonlyoccurs in individuals with iron overload and hepatitis C virusinfection; and (3) irregular iron distribution in the liver can leadto possible false-negative results.112

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In recent years, magnetic resonance imaging (MRI) tech-niques for assessing iron loading in the liver and heart haveimproved.113–115 T2 and T2* parameters have been validated forliver iron concentration. Cardiac T2* is reproducible, is appli-cable between different scanners, correlates with cardiac func-tion, and relates to tissue iron concentration.113,114 Clinicalutility of T2* in monitoring individuals with siderotic cardio-myopathy has been demonstrated.116 Calibration of T2* in theheart will be available in the near future.

Magnetic biosusceptometry (SQUID), which gives a reliablemeasurement of hepatic iron concentration, is another option117;however, magnetic susceptometry is currently available only ina limited number of centers worldwide.

Two other chelators have been introduced intoclinical use: deferiprone and deferasirox

Deferiprone (L-1), a bidentated oral chelator, available forseveral years in many countries, is administered in a dose of75–100 mg/kg/day. The main side effects of deferiprone therapyinclude neutropenia, agranulocytosis, arthropathy, and gastro-intestinal symptoms that demand close monitoring.118 Recentfindings seem to exclude any correlation between deferipronetreatment and progression of liver fibrosis.119 The effect ofdeferiprone on liver iron concentration may vary among theindividuals treated. However, results from independent studiessuggest that deferiprone may be more cardioprotective thanDFO. Compared with those being treated with DFO, individualson treatment with deferiprone have better myocardial MRIpattern and less probability of developing (or worsening preex-isting) cardiac disease.120–123 These retrospective observationshave been confirmed in a prospective study.124 After manyyears of controversy, deferiprone is emerging as a useful ironchelator equivalent/alternative to DFO.125,126

Deferasirox became recently available for clinical use inpatients with thalassemia. It is effective in adults and childrenand has a defined safety profile that is clinically manageablewith appropriate monitoring. The most common treatment-re-lated adverse events are gastrointestinal disorders, skin rash,

and a mild, nonprogressive increase in serum creatinine con-centration.127,129 Postmarketing experience and several phaseIV studies will further evaluate the safety and efficacy ofdeferasirox.

New strategies of chelation using a combination of DFO anddeferiprone have been effective in individuals with severe ironoverload; toxicity was manageable.130–135 In the past few years,particular attention has been directed to the early diagnosis andtreatment of cardiac disease because of its critical role in de-termining the prognosis of individuals with beta-thalassemia.Assessment of myocardial siderosis and monitoring of cardiacfunction combined with intensification of iron chelation resultin excellent long-term prognoses.116,136

Follow-upFor individuals with thalassemia major, follow-up to monitor

the effectiveness of transfusion therapy and chelation therapyand their side effects includes the following:

Physical examination every month by a physician familiarwith the affected individual and the disease;Assessment of liver function tests (serum concentration ofalanine transaminase) every 2 months;Determination of serum ferritin concentration every 3months;Assessment of growth and development every 6 months(for pediatric patients);Annual assessment includes the following:

● Ophthalmologic and audiologic examinations;

● Complete cardiac evaluation and evaluation of thy-roid, endocrine pancreas, parathyroid, adrenal, andpituitary function (usually after 10 years);

● Liver ultrasound evaluation, determination of serumalpha-fetoprotein concentration in adults with hepati-tis C and iron overload for early detection of hepato-carcinoma; and

Fig. 5. Disease-free survival in 61 thalassemic patients (age, 1–15 years) transplanted from a familial donor at BMT UnitOspedale Regionale Microcitemico di Cagliari, Italy.

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● Bone densitometry to assess for osteoporosis in theadult.

Assessment for liver and heart iron with MRI should be ingeneral recommended after 10 years of age and repeatedaccording to the severity of iron overload, transfusion, andchelation regimes137; andRegular gallbladder echography for early detection ofcholelithiasis,70 particularly in individuals with the Gilbertsyndrome genotype (i.e., presence of the [TA]7/[TA]7

motif in the promoter of the UGT1A gene).

Bone marrow transplantationBone marrow transplantation (BMT) from an HLA-identical

sib represents an alternative to traditional transfusion and che-lation therapy. If BMT is successful, iron overload may bereduced by repeated phlebotomy, thus eliminating the need foriron chelation. The outcome of BMT is related to the pretrans-plantation clinical conditions, specifically the presence ofhepatomegaly, extent of liver fibrosis, and magnitude of ironaccumulation. In children who lack the above risk factors,disease-free survival is over 90%138 (Fig. 5). A lower survivalrate of approximately 60% is reported in individuals with allthree risk factors. Chronic graft-versus-host disease of variableseverity may occur in 5–8% of individuals. BMT from unre-lated donors has been performed on a limited number of indi-viduals with beta-thalassemia. Provided that selection of thedonor is based on stringent criteria of HLA compatibility andthat individuals have limited iron overload, results are compa-rable with those obtained when the donor is a compatible sib.139

However, because of the limited number of individuals en-rolled, further studies are needed to confirm these preliminaryfindings. Cord blood transplantation from a related donor offersa good probability of a successful cure and is associated with alow risk of graft-versus-host disease.140,141 For couples whohave already had a child with thalassemia and who undertakeprenatal diagnosis in a subsequent pregnancy, prenatal identi-fication of HLA compatibility between the affected child and anunaffected fetus allows collection of placental blood at deliveryand the option of cord blood transplantation to cure the affectedchild.142 On the other hand, in case of an affected fetus and aprevious normal child, the couple may decide to continue thepregnancy and pursue BMT later, using the normal child as thedonor.

Therapies under investigationNew chelation strategies, including the combination or alter-

nate treatment with the available chelators, are under investiga-tion. Induction of HbF synthesis can reduce the severity ofbeta-thalassemia by improving the imbalance between alphaand nonalpha globin chains. Several pharmacologic compoundsincluding 5-azacytidine, decytabine, and butyrate derivativeshave had disappointing results in clinical trials.143 These agentsinduce HbF by different mechanisms that are not yet welldefined. Their potential in the management of beta-thalassemiasyndromes is still under investigation.

The studies on BCL11 pave the way to develop a therapybased on down-regulation of this gene, which may lead to anincrease of HbF level, thereby ameliorating the clinical severityof the disease.39 Interesting possibility of a new therapy areopen by the finding of association of low cMYB level (one ofthe gene mapping on 6q23) and high HbF level.144

The efficacy of hydroxyurea treatment in individuals withthalassemia is still unclear. Hydroxyurea is used in persons withthalassemia intermedia to reduce extramedullary masses, to

increase Hb levels, and, in some cases, to improve leg ulcers. Agood response, correlated with particular polymorphisms in thebeta globin cluster (i.e., C3T at �158 G-gamma) has beenreported in individuals with transfusion dependence.145,146

However, controlled and randomized studies are warranted toestablish the role of hydroxyurea in the management of thalas-semia syndromes.

The possibility of correction of the molecular defect in he-matopoietic stem cells by transfer of a normal gene via asuitable vector or by homologous recombination is being ac-tively investigated.147 The most promising results in the mousemodel have been obtained with lentiviral vectors.148,149 Specif-ically, the vector developed by Sadelain et al. containing HS2,3, and 4 from LCR associated with an extended beta globin geneled to partial correction of anemia in a beta-thalassemiamouse.147 Regarding the alternative approach, in a mouse modelof sickle cell anemia, Chang et al.150 were able to correct themolecular defect by homologous recombination by transfectingthe embryonic stem cells from the affected mice with a DNAfragment containing the normal beta globin gene sequences.Hematopoietic stem cells, derived from the corrected embryonicstem cells ex vivo, were able to produce HbA and HbS, therebyleading to a phenotype similar to human sickle cell trait. Thisand other similar experiments indicate the possibility of curinginherited hemoglobinopathies by homologous recombination inembryonic stem cells.

A new opportunity in the field of stem cell research has beenrecently determined by the discovery that transfection of fourtranscription factors (Oct 3–4, Sox 2, cMyc, and Klf4) inseveral types of adult cells (e.g., fibroblasts) may produce a cellwith very similar characteristics to embryonic stem cells (in-duced pluripotent stem cell [iPS]).151,152 Further research in thisfield demonstrated that cMyc may not be necessary, therebyreducing the risk of oncogenic transformation. iPS from patientsaffected by a number of monogenic diseases, such as spinalmuscular atrophy or amyotrophic lateral sclerosis, have beenalready produced. Based on this technology, Hanna et al.153

have recently obtained iPS from fibroblasts of a mouse affectedby sickle cell anemia. The iPSs were corrected ex vivo byhomologous recombination. Hematopoietic stem cells de-rived from ex vivo-corrected iPS were able to cure theaffected mouse. This experiment offers proof of principlethat genetically corrected iPS cells may lead to a cure for theinherited hemoglobinopathies.

ACKNOWLEDGMENTSThis study was supported by Grants from L.R.7 2007 Re-

gione Autonoma Sardegna.

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